Overlapping evolutionary affinities revealed by comparison of amio acid compositions ( mutation / recombination / oligonucleotide substitution / gene conversion / enzymes )

Comparison of the amino acid compositions of purified proteins indicates the presence of overlapping evolutionary affinities among enzymes of the 3-ketoadipate pathway. Isofunctional enzymes from different bacterial genera share a common evolutionary origin. Moreover, enzymes that mediate isofunctional or chemically analogous reactions within an organism appear to be evolutionarily homologous. Most remarkably, closely similar amino acid compositions are found in enzymes that mediate the following consecutive metabolic steps: lactonization, decarboxylation, hydrolysis, and transfer of a thioester bond. We have reported similar amino acid sequences in the primary structures of enzymes that mediate catalytically diverse reactions in the 13-ketoadipate pathway (refs. 1-5; Fig. 1). The inference drawn from these findings was that a novel form of mutation, the substitution of oligonucleotide sequences from one gene into another, took place as different genes for the pathway coevolved (6, 7). Recently, similar mechanisms (7, 8) involving the substitution of "minigene" sequences (9) into different regions of immunoglobulin genes have been proposed to account in part for antibody diversity. It is important to learn the extent to which oligonucleotide substitution mutations took place in metabolic evolution, and a comprehensive approach to this question would be comparison of the primary structures of all of the enzymes of the /3-ketoadipate pathway. Some years will pass before this evidence accumulates, but a related form of evidence, the amino acid compositions of the proteins (10), is more easily obtained and allows extensive comparisons to be made. Techniques ofcomparison tend to exaggerate differences in amino acid composition: thus, the methods may fail to reveal existing homology, but it is quite unusual for them to suggest a homology that does not exist (11). In this report we use a method of compositional comparison to explore relationships among enzymes of the &-ketoadipate pathway. The measure of relatedness, SAQ, is the sum of the square of the difference in mole fraction of the 16 common amino acids that can be readily and quantitatively determined in a protein hydrolysate. Marchalonis and Weltman (12) conducted more than 5,000 pairwise comparisons and found that a SAQ of <50 invariably reflected a structural homology that could be documented at the level of amino acid sequence. The amino acid compositions of some enzymes of the 8-ketoadipate pathway have been published (1-5), and we used described procedures (2) to determine the amino acid composition ofother enzymes of the pathway. Pairwise comparison of these compositions with those of25 unrelated proteins* revealed an SAQ of <50 in only 2 of 624 comparisons; the SAQ exceeded 100 in 93% of the comparisons. As described herein, comparisons among enzymes ofthe f-ketoadipate pathway often yielded low SAQs, suggesting far-reaching structural relationships. Isofunctional enzymes from different bacterial genera The most comprehensive compositional comparisons can be made with enzymes from two bacterial groups, the genus Acinetobacter and fluorescent Pseudomonas species. The two groups differ widely in their nutritional properties and in the G+C content of their DNA; the organisms also exercise different transcriptional controls in their regulation of the (3-ketoadipate pathway (15). Nevertheless, intergeneric comparisons indicate that identical steps in the pathway of the two groups are mediated by evolutionarily homologous enzymes. As summarized in Table 1, five of the eight possible comparisons reveal evolutionary homology as indicated by serological crossreaction or by amino acid sequence. In two instances [carboxymuconolactone decarboxylase (4-carboxymuconolactone carboxy-lyase, EC 4.1.1.44) and 1B-ketoadipate-succinate CoA-transferase (succinyl-CoA:3-oxoadipate CoA-transferase, EC 2.8.3.6); see Table 1], the conclusion of homology is strengthened by observation of an SAQ of <50. In addition, an SAQ of <50 is observed in comparison of the respective carboxymuconateand muconate-lactonizing enzymes (Table 1). Thus, the catechol oxygenases from the two bacterial genera are the only isofunctional enzymes of the pathway for which the inference of evolutionary homology cannot be drawn. Analogous or isofunctional enzymes that coevolved in a single cell line The two branches of the 3-ketoadipate pathway are chemically analogous, and isofunctional enzymes mediate terminal reactions in the pathway of Acinetobacter. These observations raised the possibility that parallel steps in the pathway are mediated by evolutionarily homologous enzymes (20), and this interpretation is favored by comparison of both the amino acid compositions and the NH2-terminal amino acid sequences ofthe decarboxylase-isomerase pair and Acinetobacter enol-lactone hydrolases I and II (Table 2). Compositional comparison of the Pseudomnas lactonizing enzymes yields an SAQ of 37 (Table 2), and this low number indicates that they share regions ofsimilar amino acid sequence. Because the amino acid compositions of the respective carboxymuconateand muconate-lactonizing enzymes ofAcinetobacter and Pseudomonas yield SAQ of <50 * Pseudomonas rubredoxin, Pseudomonas putidaredoxin, azurin, deoxyribonuclease, elastase, papain, basic trypsin inhibitor, prolactin, secretin, histone H4, K-casein, cytochrome c, ribonuclease, hemoglobin a chain, hemoglobin (3 chain, myoglobin, alcohol dehydrogenase, animal lysozyme, bacteriophage lysozyme, trypsin inhibitor, proinsulin, tobacco mosaic virus coat protein, flagellin serum, albumin, avidin, and acyl carrier protein. Amino acid compositions were reported by Dayhoff (13, 14). 3794 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Proc. Nati Acad. Sci. USA 79 (1982) 3795